Recent microscopic observation techniques target not only the form of a sample but also biological reactions in living specimens. Fluorescence observation using homo-fluorescence resonance energy transfer (hereinafter termed ‘Homo-FRET’) is known as one such observation technique.
Fluorescence resonance energy transfer (hereinafter ‘FRET’) is a phenomenon caused by two fluorescent molecules, one of which is termed a ‘donor molecule’ and the other of which is termed an ‘acceptor molecule’. The donor molecule is excited by excitation light and it transfers excitation energy to an adjacent acceptor molecule, whereby the acceptor molecule, which was in the ground state, is excited and emits fluorescence. Some additional requirements, such as the requirement that the donor molecule and acceptor molecule be sufficiently near each other (i.e., within a range of approximately 1 to 10 nm) must be satisfied in order for FRET to occur. FRET enables the organic activity of a specimen to be observed.
For example, a reagent (such as calcium ions), which reacts with a substance closely related in organic activity, can be used to observe the organic activity of a specimen through the presence, distribution, and changes in the calcium ions, since such a reagent changes the distance between the donor and acceptor molecules.
When the donor and acceptor molecules are different types of fluorescent molecules, the FRET is termed “Hetero-FRET.” On the other hand, when the donor and acceptor molecules are the same type of fluorescent molecule, the FRET is termed “Homo-FRET.” “Hetero-FRET” and “Homo-FRET” differ significantly from each other with regard to the observation technique used.
In Hetero-FRET, the energy transfer occurs between fluorescent molecules having a different structure and thus the fluorescent emissions from the two types of molecules have different spectrums. Thus, in Hetero-FRET, the fluorescent wavelength of the acceptor molecules is detected instead of the fluorescent wavelength of the donor molecules. The fluorescence of the donor molecules is distinguished from the fluorescence of the acceptor molecules by being at a different wavelength. Moreover, in Hetero-FRET observations, there is also a difference in the intensity of fluorescence emitted by the two types of molecules, and thus Hetero-FRET can also examine the different fluorescent emission intensities.
In Homo-FRET, the donor molecule and the acceptor molecule are identical (i.e., they are the same type of molecule) and thus the fluorescence from each has the same identical wavelength. Thus, Homo-FRET observations that use a difference in wavelength are not possible. Instead, a polarization anisotropy is used to observe Homo-FRET observations. When a fluorescent molecule is excited by a linearly polarized excitation light, the emitted fluorescence will normally have the same linear polarization. However, if FRET occurs between the excitation and the emission of fluorescence, the polarization will be disrupted so as to result in a polarization anistropy. Thus, a Homo-FRET observation can be obtained by observing the polarization anistropy that occurs in the fluorescence that is emitted when a linearly polarized light beam is incident on the specimen.
Generally, in polarization anisotropy observations, two linearly polarized light beams, one that is polarized parallel to the linearly polarized light that is incident onto the sample, and the other that is polarized perpendicular to the linearly polarized light that is incident onto the sample, are observed and their ratio is used to determine the degree of disruption of the polarization (i.e., the degree of polarization anisotrophy). In prior art polarization anisotropy observations, a polarizing beam splitter is provided in the optical path of the fluorescence in order to separate the light for observation into two beams that are polarized in orthogonal directions.
However, a high signal-to-noise ratio (hereinafter S/N ratio) is required for Homo-FRET polarization anisotropy observations. Therefore, the prior art structure, such as the structure disclosed in The Biophysical Journal, Volume 80, pp 3000-3008, June 2001, does not allow for satisfactory observations to be obtained. Furthermore, laser scanning microscopy (hereinafter LSM) requires the detection of the polarization anisotropy with a particularly high sensitivity and accuracy.